The proposed research aims to study regulatory mechanisms of transport of proteins and nucleic acids through plantintercellular connections, the plasmodesmata (PD). PD interconnect most cells within a mature plant and are critical for maintaining and regulating communication within and between different plant tissues. To study the controlling mechanisms of PD transport, this project will exploit plant viruses, pirates of plasmodesmata, that move cell-to-cell and systemically, throughout the host via these channels. Tobacco mosaic virus, one of the best studied plant viruses, moves through PD with the help of its movement protein (MP). The proposed research will utilize MP as a molecular tool to achieve three specific aims, each of which seeks to study a different aspect of PD regulation, and which together will contribute toward a single goal of understanding of the molecular mechanisms that control PD transport: (1) Study of negative regulation of cell-to-cell PD transport: the role of phosphorylation. A cell wall-associated MP kinase was isolated, that acts as a negative regulator of MP activity. The effects of phosphorylation on MP targeting to PD and on the u-helical and protease-resistant domains of MP, thought to be involved in its PD-gating activities, will be examined. Also, cellular substrates for the MP kinase will be identified and initially characterized, and reverse genetics will be used to study the functional role of MP kinase in healthy and virus-infected plants. (2) Study of positive regulation of and/or requirements for cell-to-cell PD transport: plant mutants in genes required for transport of MP. Transgenic plants were generated that express an MP-reporter enzyme fusion only in their trichomes (leaf hairs); because MP moves between cells, however, this fusion protein is found in large sectors of cells surrounding each trichome. Following mutagenesis of these plants, a mutant line was identified, in which cell-to-cell movement of MP is blocked. This mutation, which presumably affected a gene that positively regulates and/or is required for cell-to-cell PD transport, will be characterized and cloned and additional genes required for the MP transport pathway will be identified in the continuation of this genetic screen. (3) Study of regulation of systemic PD transport. We have identified an inducible plant gene that controls systemic but not local transport of viruses and post transcriptional gene silencing (PTGS) signals. The protein product of this gene will be characterized in respect to its intracellular localization, effects on PD permeability, and possible cellular partners.